Gastroenterology

Gastroenterology

Volume 150, Issue 1, January 2016, Pages 194-205
Gastroenterology

Original Research
Full Report: Basic and Translational—Liver
Interferon-γ and Tumor Necrosis Factor-α Produced by T Cells Reduce the HBV Persistence Form, cccDNA, Without Cytolysis

https://doi.org/10.1053/j.gastro.2015.09.026Get rights and content

Background & Aims

Viral clearance involves immune cell cytolysis of infected cells. However, studies of hepatitis B virus (HBV) infection in chimpanzees have indicated that cytokines released by T cells also can promote viral clearance via noncytolytic processes. We investigated the noncytolytic mechanisms by which T cells eliminate HBV from infected hepatocytes.

Methods

We performed a cytokine enzyme-linked immunosorbent assay of serum samples from patients with acute and chronic hepatitis B. Liver biopsy specimens were analyzed by in situ hybridization. HepG2-H1.3 cells, HBV-infected HepaRG cells, and primary human hepatocytes were incubated with interferon-γ (IFNγ) or tumor necrosis factor-α (TNF-α), or co-cultured with T cells. We measured markers of HBV replication, including the covalently closed circular DNA (cccDNA).

Results

Levels of IFNγ and TNF-α were increased in serum samples from patients with acute vs chronic hepatitis B and controls. In human hepatocytes with stably replicating HBV, as well as in HBV-infected primary human hepatocytes or HepaRG cells, IFNγ and TNF-α each induced deamination of cccDNA and interfered with its stability; their effects were additive. HBV-specific T cells, through secretion of IFNγ and TNF-α, inhibited HBV replication and reduced cccDNA in infected cells without the direct contact required for cytolysis. Blocking IFNγ and TNF-α after T-cell stimulation prevented the loss of cccDNA. Deprivation of cccDNA required activation of nuclear APOBEC3 deaminases by the cytokines. In liver biopsy specimens from patients with acute hepatitis B, but not chronic hepatitis B or controls, hepatocytes expressed APOBEC3A and APOBEC3B.

Conclusions

IFNγ and TNF-α, produced by T cells, reduce levels of HBV cccDNA in hepatocytes by inducing deamination and subsequent cccDNA decay.

Section snippets

Human Samples

Serum samples of 8 acute, 11 chronic hepatitis B patients, 3 healthy subjects, and 1 fulminant acute hepatitis B patient were collected and fulminant hepatitis B and nonhepatitis control liver biopsy specimens were obtained from the University Hospital Rechts der Isar. Use of human samples was approved by the local ethics committee. For detection of IFNγ and TNF-α from human serum or cell culture supernatant, human IFNγ MAX (430103; Biolegend, San Diego, CA) and human TNF enzyme-linked

Acute Hepatitis B Patients Have Increased Serum IFNγ and TNF-α Levels

It has been suggested that cytokines, particularly IFNγ and TNF-α, are involved in the clearance of HBV in acute, self-limiting hepatitis B in animal models.2, 7, 8 To investigate whether serum cytokine profiles differ between hepatitis B patients and healthy donors, we collected serum from patients of similar demographic backgrounds with acute (n = 9) or chronic hepatitis B infections (n = 11) (Supplementary Table 2). Both groups had similar HBV-DNA levels but, as expected, patients with acute

Discussion

Clearance of HBV infection by the immune system seems to require killing of infected cells by virus-specific CTL. To which extent and by which mechanisms noncytolytic T-cell function contributes to virus clearance has not been defined yet. This study shows that T cells can induce the loss of HBV cccDNA in HBV-infected, differentiated hepatocytes mainly through secretion of the cytokines IFNγ and TNF-α, which induce A3A and A3B, which play an essential role in deamination of nuclear HBV DNA and

Acknowledgments

The authors would like to thank Kathrin Kappes, Romina Bester, Theresa Asen, Raindy Tedjokusumo, Martin Feuerherd, and Olga Seelbach for their excellent technical support, and Oliver Quitt and Stephanie Kadow for characterization of Huh7-S and HepG2-H1.3 cell lines, respectively. The authors thank Christoph Seeger for providing the cell line HepAD38 and Achim Weber for support with tissue staining. The authors acknowledge the support of the nonprofit foundation Human Tissue and Cell Research,

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    Current address of Y.X.: Liver Diseases Branch, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland.

    Current address of J.L.: INSERM 1052, CNRS UMR 5286, Cancer Research Center of Lyon, University of Lyon, Lyon, France.

    Conflicts of interest The authors disclose no conflicts.

    Funding Supported in part by the German Research Foundation (grant CRC/TR 36), the German Center for Infection Research, and the Helmholtz-Alberta Initiative on Infectious Disease Research (HAI-IDR).

    Author names in bold designate shared co-first authorship.

    Authors share co-first authorship.

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